PROJECT ABSTRACT: This project is driven by the urgent need for new technology that can diagnose and follow the treatment of human skeletal muscles at the cellular level. Movement impairments due to neuromuscular disease or injury are a major cause of debility in the United States. But while muscle biopsies are the gold standard that scientists use to understand neuromuscular function and that, in addition to genomic sequence, physicians use to diagnose neuromuscular diseases, biopsies are highly invasive, painful, expensive, and relatively unavailable, and a tremendous amount of laboratory tissue processing is required to adequately understand the results. Further, biopsies can typically only be obtained at a single time point, making it difficult if not impossible to quantify disease progression or to determine therapeutic efficacy. It would be revolutionary to diagnose and treat neuromuscular disease using a device that rapidly and noninvasively measures muscle properties at the cellular level?in other words, that functions as a virtual muscle biopsy (VBx). In response to RFA-AR-19-013 (Research Innovations for Scientific Knowledge (RISK) for Musculoskeletal Diseases) we propose development of an instrument platform that exploits recent advances in photonics to create a device that noninvasively provides micron spatial resolution and kHz time resolution of skeletal muscle structure and function. To accomplish this goal, we propose the following three aims: Specific Aim 1 (R61): Develop an optical frequency comb (OFC) source with signal-to-noise ratio exceeding 40 dB. An optical frequency comb (OFC) laser source that uses thousands of laser wavelengths simultaneously represents an emerging technology that offers tremendous potential to revolutionize photonics applications inside and outside of biomedical science. Specific Aim 2 (R61): Create and validate the photonic system necessary to interrogate muscle across the skin. An optical bandwidth greater than 350 nm is necessary to accommodate the complexity of muscle structures and perform a VBx. We hypothesize that VBx can noninvasively measure sarcomere length, fiber size, fiber type, and indicators of fibrosis and denervation with high resolution in real time. In a rat model, we will directly compare data obtained noninvasively using VBx with data obtained by invasive manual tissue processing of the same muscles to evaluate the accuracy and fidelity of VBx. Specific Aim 3 (R33): To perform serial transdermal sarcomere length measurement in patients with wrist flexion contractures. As the first proof-of-principal experiment in humans, we will use VBx to measure a known entity, sarcomere length, in children with cerebral palsy who have wrist flexion contractures just prior to surgery and validate these data against the same muscle sampled intraoperatively. We believe that this device can revolutionize our understanding of neuromuscular function, permit objective evaluation of therapy, and provide a real-time three-dimensional image of biological tissue.